This application addresses several important issues for understanding the role of Munc13 proteins as tethering/priming factors for membrane fusion. At multiple stations in the secretory and endosomal trafficking pathways, vesicle-target membrane interactions are mediated by a diverse group of tethering factors. These factors recognize vesicle components and target membrane constituents to achieve vectorial targeting. Tethering factors also actively engage SNARE proteins to assemble trans SNARE complexes for fusion. In the regulated secretory pathway of neuroendocrine cells, vesicle priming involves SNARE complex assembly and requires Munc13 family proteins. Munc13 family proteins have sequence similarity in SNARE-binding domains that are homologous to tethering factors. We found that three members of the Munc13 protein family (Munc13- 1, Munc13-4, CAPS) exhibit similar SNARE-binding properties and similarly promote SNARE complex formation and SNARE-dependent liposome fusion. Surprisingly, ubMunc13-2 and CAPS-1 functioned non- redundantly and cooperatively in evoked DCV (dense-core vesicle) exocytosis in neuroendocrine PC12 cells. This indicates that each of these proteins is distinct in its mechanism for assembling SNARE complexes. We will determine the basis for these differences using a series of novel assays for SNARE complex assembly in Aim 1. The assays take advantage of our success in generating full-length active proteins for biochemical studies. Completion of this work will provide a new view of SNARE regulatory proteins, significantly impacting our understanding of the molecular basis for neuropeptide and peptide hormone secretion. We have found that two other proteins in the Munc13 family, Munc13-4 and BAIAP3, act as tethering/priming factors for intracellular membrane fusion events. Ubiquitously-expressed Munc13-4 was required for the merge of a subset of endosomal compartments. In neuroendocrine cells, Munc13-4+ endosomes also interacted with DCVs in a novel process that will be elucidated in Aim 2. Neural/endocrine- specific BAIAP3 is required for evoked peptide secretion in neuroendocrine cells but localizes to the late Golgi and to a small subset of DCVs. In Aim 3, we will determine if BAIAP3 functions in the homotypic fusion events that characterize DCV maturation. For both Munc13-4 and BAIAP3, we will determine their sites of action, define the fusion events they promote, and identify the SNARE proteins they regulate. This work will characterize intracellular membrane fusion events that currently have no known regulators, and will reveal novel points of intersection between the endosomal and regulated secretory pathways in neuroendocrine cells. It will also impact the understanding of pathologies linked to mutations in Munc13-4 and BAIAP3.